Electrophoresis is one of the most widely used methods for separation and purification of charged biological species. Proteins, DNA, other chemicals, cellular substructures, organelles and even whole cells have been separated by electrophoresis on an analytical, and in some cases, preparative scale. This widespread application results from the essentially universal characteristic of biological components and chemicals to acquire a charge in polar or ionic solution through ionization or ion adsorption. These charged species can then be separated from one another based on the relative differences between their migration velocities in electric field. Virtually anything that will dissolve or suspend in a conductive fluid may be seperated. Popularity electrophoresis is also due to its ability to discriminate between even closely related species. For example, analytical two dimensional electrophoresis has been shown to resolve up to 5,000 separate proteins in a single sample. This ability to separate a wide range of compounds with high selectivity suggests that a large-scale electrophoretic method would be an extremely valuable technique in downstream bioprocessing.
Promise of a powerful, relatively robust and simple bioseparation unit operation has directed many efforts towards development of large-scale methods. Thus electrophoresis could be used not only for analysis of compounds but as a large scale purification and separation device for biological species. Several fundamental characteristics of the electrophoretic process, however, have precluded the simple scale-up of batch analytical techniques. These include the Joule heating effect of the electric current, the complication of electroosmotic flow, and the dominant effect of convective dispersion in instruments larger than a critical size. The principle analytical anticonvective packings, such as porous gel, also impede heat dissipation in larger devices, limiting throughput and cannot be used for separation of many larger species, such as whole cells and organelles.
In addition, batch operation inherently reduces the possible capacity of such apparatuses. Research into large-scale electrophoresis has therefore centered almost exclusively on continuous devices utilizing no discontinuous anticonvective packing. Many such continuous devices have been proposed over the last thirty-five years. These include models with essentially no packing, freeflow) devices and other devices using packing.
One such continuous electrophoresis device is U.S. Pat. No. 4,642,169 (the Iowa Electrophoresis Column--IEC). This invention provides a continuous rotating annular electrophoresis column for separation. The electric field for this device is applied in the axial direction combined with a forced axial eluate flow through a slowly rotating annular column which is filled with anti-convective packing. By rotating the column the product path appears as helical bands each with a characteristic stationary exit point at some angular coordinate at the bottom of the column. The packing consists of spherical glass micro beads with the eluent and sample filtrating through the interstitial spaces between the beads on the basis of their electric charge. Despite this advancement in providing improved throughout the device is still plagued with the characteristic problems of packing. Traditional packing which includes gels such as polyacrylimide gel, and glass beads encounter several dispersive problems affecting the efficiency of the system for large scale separation. Thermal gradients across the device, convective dispersion due to micromixing, stream splitting and union, diffusion normal to the bulk velocity axis and effects of the device walls are all problems encountered with traditional packings.
In all current large scale electrophoresis designs, neat generated by Joule (electrical) heating is removed through one or more outer walls of the device. This results in a significant temperature gradient between the warm center of the apparatus (mid radius of an annular design such as the IEC) and the cool wall. This temperature gradient in turn creates liquid buoyancy and viscosity, voltage and chemical potential gradients across the device. In the vertical downflowing orientation involved in continuous flow designs, the difference in liquid buoyancy means that the warmer lighter eluent at the center will flow downward slower than the solvent near the wall. This results in product dispersion due to the distribution of bulk fluid velocity across the device, and can lead to destructive thermal convection. The viscosity gradient only complicates the variance in fluid velocity. The voltage gradient across the device means that the electrophoretic force and thus the electrophoretic velocity exerted on the molecule also varies across the device resulting in additional dispersion. Similarly the electroosmotic flow varies with the voltage gradient. Current attempts to alleviate thermal gradients in the electrophoresis involve circulation of a coolant around the walls of the device, in an attempt to cool and eliminate convection.
While conventional anticonvective packing, such as spherical glass beads, reduces large scale convective mixing, convective dispersion still remains. Fluid streamlines separate and mix as they pass around or through the packing, and product molecules diffuse between streamlines of different velocity and stagnant zones close to or in the packing. The pores created by interstitial spaces in the conventional packing; whether actual pores in a continuous packing, such as a gel, or whether interstitial spaces between discontinuous particles, meander through the packing and facilitate the dispersion.
And finally, due to the attempt to cool the device, the presence of device walls often leads to variation in electrophoretic environment besides those caused by thermal gradients between the vicinity of those walls and the "center of the apparatus". For example in discontinuous packing the interstitial void space increases near the walls leading to an increase in eluent velocity compared to more central regions.
It is an object of the present invention to provide a type of electrophoresis packing and cooling system which may be used with both continuous flow and batch large scale electrophoresis techniques. The system comprises a bundle of capillaries for flow of sample, which may be cooled by coolant circulated immediately around the exterior of the capillaries.
It is a further object of the invention to provide a packing for electrophoresis which will reduce and/or eliminate thermal gradients across the electrophoresis column by allowing a cooling fluid to be circulated within the column immediately around the exterior of the capillaries.
A further object of the invention is to provide a design for an electrophoresis device which will eliminate convective dispersion, radial diffusion, variations in electropotential gradient and wall effects of earlier type packing such as discontinuous packing.
A still further object of the invention is to provide an electrophoresis large scale separation device which will not be limited to merely batch separation but will also be useful for continuous flow separation.
A further object of the invention is to provide a multiple capillary bed as a replacement for discontinuous anticonvective packing and for continuous packing such as gels.
These and other objects of the invention will be more clear in the description of the invention which follows.